There is a pressing need for a technology platform that can aid rapid AST profiling where MIC results are presented within a narrow time window of less than 3 hours. This acquires tremendous significance during bloodstream infections and rapid onset sepsis; the choice of antibiotics to surmount emergent antibiotic resistance and associated treatment modality in reduction of host inflammatory response is crucial for patient survival and recovery. Such a platform will finally usher in the Precision medicine approach for treating bacterial infections, whereby physicians are able to track antibiotic resistance profiles f pathogens in real time and adjust their antibiotic dosing regimens. It is highly unlikely that culture techniques can still be improved to significantly decrease total assay time (TAT). Therefore, this proposed system focuses on improving TAT by using molecular technologies for identification and characterization of microorganism susceptibility profile directly from fresh whole blood specimen of BSI patients. A first step forward, to decrease time-to-result, can be achieved by utilizing blood lysis centrifugation. Moreover, eliminating steps for obtaining clinica isolates will lead to the most optimal turnaround time. The overall hypotheses of this application are that a CentriFluidic system can be used to perform a rapid PID and AST directly from whole blood without a need of blood cultivation or PCR amplification based on our successful clinical feasibility studies on urinary tract infection (UTI) directly from raw urine specimen with reproducible demonstration of polymicrobial infection detection and multi-drug resistance profiling. We developed, demonstrated and published an innovative molecular-based genotypic-phenotypic-hybrid approach for multiplexed bacterial PID and AST profiling with 100% clinical sensitivity, 96% clinical specificity, 98% minimum inhibitory concentration (MIC) and 97% categorical agreement in our most recent ongoing clinical feasibility study on 73 raw clinical urine samples. However, critical limitations of the current UTI platform exist, including inabilityto detect much lower abundance of bloodborne pathogens, the lack of system integration with an ultracentrifugation module and inability to cover all clinical relevant pathogens for BSIs. The main goal of this RO1 research project is to combine the advantages of lab automation, rapid molecular analysis, melt-curve signature analysis, genotypic pathogen quantification and phenotypic antibiotic conditions to dramatically improve the sensitivity and specificity of rapid, evidence-based PID and AST directly from patient blood samples. We propose to test the following hypotheses: Specific Aim 1: Transition the current electrochemical-based molecular analysis PID/AST platform technology from UTI to BSIs - Hypothesis: Lysis centrifugation can address the change of matrix effect from raw urine to whole blood samples and the issue of low abundant pathogen for BSIs. Specific Aim 2: Develop a dual-mode electrochemical-based dynamic hybridization analysis algorithm to expand the BSI PID/AST panel - Hypothesis: Dynamic hybridization analysis can be utilized to expand the species-specific identification of common and emerging pathogens for BSIs. Specific Aim 3: Prototype, validate and manufacture the CentriFluidic cartridge and system - Hypothesis: Ultracentrifugation (up to 50,000 g, gravitational force) can be incorporated into a multiplexed fluidic cartridge for a fully automated BSIs PID/AST from whole blood samples in 3 hours. Specific Aim 4: Clinically validate the rapid BSI PID/AST CentriFluidic system according to CLSI guidelines - Hypothesis: Blood samples spiked with ATCC strain bacteria used in the analytical validation studies represent critical matrix characteristics of fresh whole blood samples from patients.